Mass spectrometers have become one of the essential tools of the biochemistry lab. Biochemists take advantage of the capabilities of mass spectrometers to determine molecular weights of biomolecules, monitor bioreactions, detect post-translational modifications, perform protein, and oligonucleotide sequencing, and many more applications. During the past decade, one of the methods, which became most successful for the mass spectrometric analysis and investigation of large molecules is a method known as MALDI (Matrix-Assisted Laser Desorption Ionization). This method, which in application to time-of-flight (TOF) mass spectrometry (MS) is known as MALDI-TOF MS, is a relatively novel technique in which a co-precipitate of an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. The sample (analyte) is suspended or dissolved in a matrix.
Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. Matrices are small organic compounds that are co-crystallized with the analyte. It seems that the presence of the matrix spares the analyte from degradation, resulting in the detection of intact molecules as large as 1 million Da (Dalton or amu are atomic mass units used in microbiology). For example, in time-of-flight mass spectrometers, the ionized biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass-to-charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides, and oligonucleotides, with molecular masses, e.g., between 200 and 350,000 Da.
Another advantage of MALDI in application to mass spectrometry is that this method allows for vaporization and ionization of non-volatile biological samples from a solid-state phase directly into the gas phase.
The most important step in MALDI is sample preparation. During this step, the matrix and analyte are mixed, and the mixture is dried on a probe or, as it is more common now, on a sample plate. Upon preparation, the sample is loaded into the mass spectrometer.
A laser beam serves as the desorption and ionization source in MALDI. The matrix plays a key role in this technique by absorbing the laser light energy and causing part of the illuminated substrate to vaporize. A rapidly expanding matrix plume carries some of the analyte into the vacuum with it and aids the sample ionization process. The matrix molecules absorb most of the incident laser energy minimizing sample damage and ion fragmentation (i.e., soft ionization). Nitrogen lasers operating at 337 nm (a wavelength that is well absorbed by most UV matrices) are the most common illumination sources because they are inexpensive and offer the ideal combination of power/wavelength/pulsewidth. However, other UV and even IR pulsed lasers have been used with properly selected matrices.
Once the sample molecules are vaporized and ionized, they are transferred into a mass spectrometer where they are separated from the matrix ions and individually detected.
A sample support used in the aforementioned system may comprise a thin, substantially square plate of stainless steel or another suitable material, e.g., approximately 1.5 mm thick and 50 mm wide sample. An example of a sample support geometry is the one described, e.g., in U.S. Reissued Patent RE 37,485 filed by Marvin L. Vestal and published on Dec. 25, 2001. The system is equipped with a support transport mechanism working in vacuum and intended for automatically inputting and outputting each of the sample supports into and from the sample receiving chamber of the mass spectrometer.
The sample plate carrier described in the aforementioned earlier inventions may contain precisely located holes to allow the position and orientation of the plate to be accurately determined relative to a moveable stage, which is required both in the sample loading step and in the ion source of the mass spectrometer. The sample plate also contains a plurality of precisely determinable sample positions on the upper sample-receiving surface of the plate. The sample plate may thus contain 100 sample positions each identified by a sample spot, which is about 2.5 mm in diameter in a precisely known location on the plate, with each sample support being suitable for accepting a few microliters of sample solution.
The sample support is rigidly attached to a ferromagnetic material handle, which is used to engage an electromagnetic device for the purpose of transporting the sample plate between component systems. The sample plate has two or more precisely located holes which locate the sample holder when installed in the sample receiving stage in the ion source of the mass spectrometer and in the sample transport trays.
Another example of a similar sample plate support or holder intended for atmospheric-pressure MALDI can be found in co-pending U.S. patent application Ser. No. 10/615,733 of the same applicant. The sample plate carriers of this application are designed for automatic loading/unloading into and from the sample storage device, such as a sample plate carrier cassette which is used in conjunction with a computer-controlled sample holder handling mechanism for taking a sample plate carrier from the aforementioned cassette, extracting the sample plate from the carrier, inserting the sample plates to the target flange for interface with a mass spectrometer orifice, securing it in a working position for analysis, unloading back to the sample plate carrier after completion of the analysis, and inserting the sample plate to a desired cell of the sample storage device.
In the specific embodiments of aforementioned U.S. patent application Ser. No. 10/615,733, the system is provided with two holder transportation units. The sample holder or carrier of the aforementioned type has specifically shaped slots for engagement with grippers of a mechanism intended for extraction of the sample plate carriers from the cassette and for loading them into the cassette and for disconnection of the sample plates from the respective carriers. Information about individual positions of the holders is stored in the memory of a common central processing unit, which also controls operation of all actuating mechanisms of the aforementioned modules. In other words, the aforementioned carriers only hold the sample plates with multiple sample cells and do not carry any address information or data about the samples or sample plate carriers themselves or about the analysis history, etc.
If analysis is relatively low in volume, it is common that the aforementioned information is loaded manually to a mass spectrometer and to the central processing unit for handling the sample plate carriers. However, when a large number of samples is to be analyzed with the use of automatic loading/unloading devices such as industrial robots or the transportation system of the type described in the aforementioned patent application, it becomes difficult to analyze different samples by different methods, as well as to keep the correct data regarding the sample history and location of various sample plates in the cells of the sample storage device. It is also difficult to keep information on the exact location of the samples on the respective sample plates.
Attempts have been made to solve the above problems by providing the sample holders with a permanent bar code, which may simplify tracking between the sample and the generated data. For example, U.S. Pat. No. 6,064,754 issued on May 16, 2000 to R. Parekh, et al. discloses a computer-assisted methods and apparatus for identification and characterization of biomolecules in a biological sample. It is stated in the aforementioned patent that methods of indexing the information record to the proper sample can include the assignment of matching numbers to the record and the sample. This process is preferably automated through the use of barcodes and a barcode scanner. As each sample is processed, the scanner is used to record the sample identification number into the memory, which tracks the sample through its various manipulations, thus preserving the link between record and sample. The use of barcodes also permits automated archiving and retrieval of stored samples. The barcodes can be engraved on the sample plate carriers permanently.
However, using this approach, it is still possible to misalign data because the sample plate carriers can be reusable and the holders with the same permanent bar codes could be used for carrying different samples for analysis at different time.
In another approach, a removable bar code sticker attached to the sample plate can be used to uniquely identify the plate holder for specific sample plate and for specific type of analysis. However, it may be more difficult to clean such plate holders for reusing. Furthermore, an undesired situation may occur when the bar code sticker is separated from the holder during handling. Another disadvantage is that for reuse of the holder the old sticker has to be removed and replaced by a new one. This requires additional time. Some companies offer labeling machines specially for labeling the microbiological sample plate carriers with barcode stickers. For example, Finnish company Oy Ideos, Ltd. produces state-of-the-art identification and data collection system. When the sample plate carrier with the barcode sticker arrives at the laboratory, all the information associated with the sample is read and automatically entered into the laboratory information system with the use of a special data-reading unit supplied by the aforementioned company. However, the use of automatic barcode reading devices does not solve the problems associated with barcodes in general.
Another disadvantage of the barcoded sample plate carriers is that they are difficult to protect from contamination and cross-contamination during handling by the robotic systems or other mechanical grippers.
The aforementioned Finnish company Oy Ideos, Ltd. also produces manually handled carriers for blood analysis (phlebotomy) samples. All appropriate information is sent to the laboratory by the re-usable sample plate carriers that will replace requests, which normally have been written on paper. The data is written on memory built into an RF ID board. Compared with the traditional technologies (such as barcodes) used for phlebotomy data transfer, the application of the RF ID technology offers such feature as possibility to add and modify data on the fly so that information can be updated at any point of time during the sample handling process.
Although the aforementioned system of manually-handled carriers with a built-in memory is efficient and convenient due to wireless access to the memory of the carrier for reading/wring the information, the carries of the aforementioned type use RF communication and should have relatively large dimensions. They are intended for manual transportation, handling, loading, and unloading into a blood-analysis control system installed in a laboratory. In other words, the phlebotomic sample plate carriers of Oy Ideos, Ltd. are inapplicable for genomic studies, where thousands of samples have to be treated during a short period of time on a series of sequentially arranged units of biomedical analytical equipment. The sample plate carrier with transmission and receiving of RF signals should have large overall dimensions. Furthermore, radio frequency signals generated by the cards may interfere with sensitive instruments of the analytical laboratory, to say nothing of the case if such carries were installed side by side into the cells of the sample plate carrier cassette where it would be impossible to identify the required carrier without activation of all of them at the same time.
Thus, sample plate carriers with on-rout information about the samples and history of processing suitable for high throughput analysis of high-volume samples, such as those required, e.g., for genomic MALDI mass-spectrometric study, are still unknown.